Anais da Academia Brasileira de Ciências (2019) 91(1): e20180358 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201920180358 www.scielo.br/aabc | www.fb.com/aabcjournal
Saline extract from Malpighia emarginata DC leaves showed higher polyphenol presence, antioxidant and antifungal activity and promoted cell proliferation in mice splenocytes.
BÁRBARA R.S. BARROS1, BRUNO RAFAEL BARBOZA1, BÁRBARA A. RAMOS2, MAIARA C. DE MOURA2, LUANA C.B.B. COELHO2, THIAGO HENRIQUE NAPOLEÃO2, MARIA TEREZA S. CORREIA2, PATRÍCIA MARIA G. PAIVA2, IRANILDO JOSÉ DA CRUZ FILHO1, TÚLIO DIEGO DA SILVA3, CLÁUDIA S.A. LIMA4 and CRISTIANE M.L. DE MELO1
1Department of Antibiotics, Biosciences Centre, Federal University of Pernambuco, Av. Prof. Artur de Sá, s/n, 50740-520 Recife, PE, Brazil 2Department of Biochemistry, Biosciences Centre, Federal University of Pernambuco, Av. Prof. Moraes Rego, 1235, 50670-901 Recife, PE, Brazil 3Center for Strategic Technologies of the Northeast, Av. Prof. Luís Freire, 1, 50740-545 Recife, PE, Brazil 4Department of Biophysics, Biosciences Centre, Federal University of Pernambuco, Av. Prof. Moraes Rego, 1235, 50670-901 Recife, PE, Brazil
Manuscript received on April 19, 2018; accepted for publication on June 21, 2018
How to cite: BARROS BRS, BARBOZA BR, RAMOS BA, MOURA MC, COELHO LCBB, NAPOLEÃO TH, CORREIA MTS, PAIVA PMG, CRUZ FILHO IJ, SILVA TD, LIMA CSA AND MELO CML. 2019. Saline extract from Malpighia emarginata DC leaves showed higher polyphenol presence, antioxidant and antifungal activity and promoted cell proliferation in mice splenocytes.. An Acad Bras Cienc 91: e20190916. DOI 10.1590/0001-3765201920180358. Abstract: Currently, the research of new natural compounds with biological potential demonstrates great ethnopharmacological importance. In this study, we evaluated the biological properties promoted by saline extract from Malpighia emarginata DC leaves, whose objective is to evaluate the antioxidant, antimicrobial and cytotoxicity potential. Phytochemical characterization was performed by UPLC-MS chromatography to identify the chemical compounds. For the antioxidant potential, DPPH, ATT and FRAP methods were used. The antibacterial and antifungal tests were performed evaluating the MIC50, MIC90, CMB and CMF parameters. Moreover, antibiofilm action was evaluated. Cytotoxicity and proliferation were performed using splenocytes from Balb/c mice and were evaluated by cytometry. We found a list of phenolic compounds among other bioactive compounds in the M. emarginata saline extract. In addition, higher antioxidant profile and antifungal activity against different strains ofCandida spp. was promoted by the saline extract. Splenocytes showed greater cell viability (more than 90%) and showed higher proliferate index in 24 and 48 hours of incubation with the extract. Saline extract from Malpighia emarginata DC has potential action like antioxidant and antifungal agent without promote animal cell damage. Key words: bioactive compounds, cytotoxicity, Malpighia emarginata DC, phytochemical.
INTRODUCTION the diversity of plant species, particularly those used
Human culture has been profoundly influenced by for therapeutic purposes. Currently, the continuous knowledge about the medicinal properties of plants Correspondence to: Cristiane Moutinho Lagos de Melo has been the focus of several scientific investigations E-mail: [email protected] ORCID: 0000-0002-8831-0163 around the world. Is important and necessary the
Biological Sciences An Acad Bras Cienc (2019) 91(1) BÁRBARA R.S. BARROS et al. Malpighia emarginata PRESENT BIOLOGICAL ACTIVITIES study of phytochemical constituents, therapeutic Many researches show important biological action mechanism and identify active principles actions promoted by different organic extracts responsible for many biological activities observed (like phenolic and ethanolic fractions) made using in medicinal plants (Lin et al. 2018). Moreover, leaves of M. emarginata (Silva 2014, Oliveira many authors have been publishing evidences 2015, Rezende et al. 2018). However, is known that about the efficacy of medicinal plants and their the true use of M. emarginata in local communities constituents on the improvement of diseases as is like teas or infusions using water as the solvent. cancer (Shirzad et al. 2011), atherosclerosis (Asgari Although the studies about M. emarginata et al. 2012) and diabetes (Asgary et al. 2011). fruit are extensive, is very important to study Malpighia emarginata DC. is a plant found the phytochemical composition and biological throughout Central and South America. It is a tropical activities promoted by other organs of this plant. specie originating in the Caribbean region, being Thus, the present study aimed investigates the known as the Antillean cherry (Oliveira et al. 2012, phytochemical composition of the saline extract Moreira et al. 2016). In Brazil the fruit is known as from Malpighia emarginata DC leaves and shows acerola and possess high content of ascorbic acid the initial biological activities promoted by this (vitamin C), carotenoids, anthocyanins, thiamine, compound. riboflavin, niacin, proteins and mineral salts, mainly iron, calcium and phosphorous, being considered a MATERIALS AND METHODS functional food (Mezquita and Vigoa 2000, Rosso VEGETABLE MATERIAL AND EXTRACT et al. 2008, Alvarez-Suarez et al. 2017). This plant PREPARATION also has other phytochemical constituents such as flavonoids, phenolic acids and polyphenols The leaves of Malpighia emarginata were collected (Bataglion et al. 2015, Malegori et al. 2017). All in the city of Recife, state of Pernambuco, Brazil. these compounds are able to minimize oxidative The botanical identification was performed by the damage caused by the reactive oxygen and nitrogen Professor Marlene Barbosa, on May 30, 2017, species in animal organism (Oliveira et al. 2012) at the Herbarium Geraldo Mariz, located at the and prevent some chronic diseases such as cancer Center of Biosciences of the Federal University (Mezadri et al. 2008, Leffa et al. 2014). Moreover, of Pernambuco through exsiccate deposited under these molecules also exhibit potential therapeutic registration number 82.615. After collection, the effect as anti-inflammatory, radiation-protective, leaves were kept at room temperature for drying chemoprotective, vasoprotective, inhibition of LDL during 4 days. The dried leaves were crushed in oxidation and decrease the risks of cardiovascular Reverse Black Oster and the pulverized material diseases (Wang et al. 1997, Seeram and Nair 2002, (20g) was diluted in 0.15 M NaCl (200 mL) in the Rosso et al. 2008). proportion of 10% (w/v). Therefore, using an orbital Local communities, especially in northeast and alternative incubator shaker (Lab Companion™ of Brazil, use the juice of acerola mainly for IS-971) the material was kept under agitation (400 respiratory diseases as flu and bronchitis. However, rpm) in constant temperature (28 ºC) for 16 hours. some accounts showed that leaves of plant were After the stirring time, the material was filtered used to make tea associated with garlic and honey using paper filter and centrifuged (15 min, 10,000 to flu symptoms. In addition, recent biological x g). The obtained supernatant was collected and studies have shown that acerola consuming is not called saline extract from Malpighia emarginata dangerous to human cells. leaves. After this, the extract was lyophilized and
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9.2 g of crude material was obtained. Material was ANALYSIS OF PHYTOCHEMICAL COMPOUNDS THROUGH OF ULTRA PERFORMANCE LIQUID stored at -20 °C until use. CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRY – UPLC-MS DETERMINATION OF TOTAL PHENOLS Ultra-performance liquid chromatography (UPLC) The total phenols were determined by the Folin- was performed with an Acquity H-Class (Waters) Ciocalteu method according to Li et al. (2008) employing a 2.1 x 100 mm BEH column with a with some modifications. Extracts diluted at 1 mg/ particle size of 1.7 μm. The column was maintained mL were dissolved in distilled water. In 0.1 mL of under a constant temperature of 40 ºC and the auto Folin solution (1:10 v/v) was added 0.02 mL of injector at 10 ºC. We use an aqueous solution the diluted extracts. After 3 minutes of incubation (eluent A) containing 2% methanol (MeOH), 5Mm (in the dark), 0.08 of sodium carbonate (7.5%) ammonium formate and 0.1% formic acid and a was added and incubated for 120 minutes in the methanol solution (eluent B) containing 0.1% dark at room temperature. After this period, the formic acid, which were pumped at a flow rate of absorbance of the samples was measured at 765 nm 0.3 mL/min. Ten microliters of the M. emarginata extract was injected. Elution was performed in against a blank (reagent added to distilled water). gradient mode and the initial condition (98% A / A calibration curve was prepared by plotting the 2% B) was maintained for 0.25 minutes. The B ratio absorbance as a function of the concentration of increased linearly to 99% in 8.5 minutes, remaining gallic acid (0 - 500 μg/mL) was prepared and the at 99% B for one minute, followed by immediate linear equation (y = 0.0048x + 0.0016 R² = 0.9999). decreased to 2% B, where it was maintained for up Phenols are expressed in gallic acid equivalent (mg to 11 minutes. The UPLC system was coupled to a EAG/g of extract). single quadrupole mass spectrometer SQ Detector 2 (Waters®). The data were obtained in fullscan DETERMINATION OF FLAVONOID CONTENT mode, analyzing masses between 100 and 1000 The determination of flavonoids followed the Da, in negative ionization. The acquisition of the methodology in accordance with Woisky and chromatograms and mass spectra was obtained through MassLynx ™ software (Waters®). Salatino (1998) and Santos et al. (2017) with some modifications. The experiment consists of pipetting EVALUATION OF ANTIOXIDANT ACTIVITIES IN 1 mL of extract (1 mg/mL) in test tube. After this, VITRO
1 mL of aluminum chloride (AlCl3, 5%) prepared Total Antioxidant Activity in methanol (2 mL) was added in tubes. The blank was prepared using 3 mL of methanol and 1 mL of The total antioxidant activity promoted by saline extract from Malpighia emarginata leaves was aluminum chloride. After 30 minutes of incubation determined as a function of ascorbic acid, according (in dark) the absorbance was measured at 425 nm. with (Pietro et al. 1999). Saline extract (100µL at A standard curve with quercetin (0 - 500 μg/mL) 500 µg/mL) was mixture with ascorbic acid (1 mg/ was performed to obtain the equation Y = 0.023x mL) and 1 mL of phosphomolybdenum solution + 0.1509, R² = 0.9956. The assays were performed (600 mM sulfuric acid, 28 mM sodium phosphate in quintuplicate. Flavonoids are expressed in and 4 mM ammonium molybdate). This mixture quercetin equivalent (mg QE/g of extract). was incubated in water at 95 °C for 90 minutes.
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After returning to room temperature, the absorbance FRAP reagent and this solution was incubated for are measured at 695 nm against a blank (1 mL of 30 minutes at 37 °C in the dark. Subsequently the solution and 0.1 mL of water). A standard curve absorbance was measured at 593 nm. A standard with ascorbic acid (0 - 500 μg/mL) was performed curve with FeSO4 (0 - 1000 μg / mL) was performed to obtain the equation Y = 0.019x + 0.0723, R² = to obtain the equation Y = 0.0024 x + 0.0019, R² = 0.9937. Assays were performed in quintuplicate. 0.9953. The results were expressed as mg EFeSO4 Total antioxidant activity was calculated by the (II)/g of extract. formula ATT (%) = [(As - Ac) / (Aaa - Ac)] 100, ANTIMICROBIAL ASSAYS where: Ac = Control Absorbance, As = Sample Absorbance and Aaa = Ascorbic Acid Absorbance. Bacterial and fungal strains, culture conditions Free radical sequestration by DPPH and preparation of samples
The antioxidant activity of saline extract was Fungal strains were obtained from Micoteca of measured by the stable radical 2,2-diphenyl-1- Mycology Department (URM) and the collection picrylhydrazyl (DPPH), as described by (Blois of microorganisms from the Department of 1958). Using 0.04 mL of different concentrations of Antibiotics (UFPEDA), both departments the saline extract (3.9; 7.8; 15.6; 31.3; 62.5; 125; 250 belonging to Biosciences Center of Federal and 500 μg/mL) it was added 0.25 mL of the DPPH University of Pernambuco. Stock cultures were kept under refrigeration (-20 °C) in sterilized solution (1 mM and OD517 = 0.650 ± 0.50). After incubation time (25 minutes) at room temperature, skimmed milk containing 10% (v/v) glycerol or protected from light, the absorbance was measured 30% (v/v) Mueller Hinton glycerol broth. Bacterial at 517 nm. We used as control the DPPH solution and fungal strains used in this study are: Candida added to distilled water. Assays were performed in albicans (URM 5901), Candida krusei (URM quintuplicate. The sequestration of DPPH radicals 6391), Candida tropicalis (URM 6551), Candida was calculated by the formula: SRL [DPPH•] parapsilosis (URM 6951), Candida glabrata (URM (%) = [(As - Ac) / Ac] x 100. When: As = sample 4246), Escherichia coli (UFPEDA 224), Klebsiella Absorbance and Ac = control Absorbance. The pneumoniae (UFPEDA 396), Pseudomonas antioxidant activity of saline extract was measured aeruginosa (UFPEDA 416), Salmonella enteritidis by the stable radical 2,2-diphenyl-1-picrylhydrazyl (UFPEDA 414), Staphylococcus aureus (UFPEDA (DPPH). 02), Staphylococcus saprophyticcus (UFPEDA 833). For antimicrobial activity assays, bacterial Reduction of Ferric Ion (FRAP assay) and fungal species were cultured in Mueller Hinton The stock solution of the FRAP assay was prepared and Sabouraud Dextrose agar medium, respectively, overnight at 36 °C, and subsequently the colonies with 300 mM acetate buffer (3.1 g CH3COONa and were resuspended in sterile saline solution 16 mL CH3COOH) at pH 3.6, 10 mM TPTZ (2.4, 6-tripyridyl-s-triazine) solubilized in 40 mM HCl, (0.15 M NaCl) and adjusted turbidimetrically at a wavelength of 600 ηm (DO ) to obtain the and 20 mM FeCl3 solution. The working solution 600 was prepared by mixing the acetate buffer, TPTZ suspension equivalent to 106 colony forming units and FeCl3 in a ratio of 10:1:1 (v/v/v) and incubated (CFU) per mL. For the assay, the samples were for 5 min at 37 ºC. Saline extract (25µL at 1 mg/ filtered on the sterile polyvinylidene difluoride mL of concentration) was added in 180 µL of (PVDF) syringe filter of 13 mm x 0.22 μm.
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Determination of Minimum Inhibitory reduce the number of CFU in 99.9% in relation to Concentration (MIC), Minimum Bactericidal the initial inoculum. Each assay was performed in Concentration (MBC) and Minimum Fungicidal triplicate using three independent experiments. Concentration (MFC) Antibiofilm Activity Analysis The minimum inhibitory concentration (MIC) Biofilm formation was evaluated by the crystal promoted by saline extract from Malpighia violet method in flat bottom polystyrene microtiter emarginata leaves was determined by the plates. Then 80 μL of Milli-Q water, 40 μL of the microtiter test proposed by the Clinical and Sabouraud Dextrose medium and 80 μL of the Laboratory Standards Institute (CLSI). In 96- fungal suspension (108 CFU/mL; sterile saline) well microtiter plates were added (80 μL) into the were added, respectively. A DO was also fourth well from which a serial dilution in sterile 600 launched using a microplate reader and as plates Milli-Q water was performed to the twelfth well of were incubated at 36 °C for 24h. After this period the same row. Subsequently, 40 μL of the medium plates were read again to determinate the bacterial Mueller Hinton broth (bacteria) or Sabouraud and/or fungal growth rate in 600 nm. A sequential Dextrose (yeast) were added to all but the first stage corresponds to the analysis of biofilm that wells, where 200 μL of the culture medium was is after sequencing of planktonic cells (unbound added, corresponding to the sterility control. The cells). In addition, the samples were washed with antibiotics ampicillin and tetracycline (8 μg/mL) 0.15 M NaCl in pre-fixed methanol for 20 min, and antifungal fluconazole (64 μg/mL) were used followed by a setting at 50 °C for 60 min and then as positive controls in the second well. Finally, the labeled with 0.4% (w/v) crystal violet for 25 min at bacterial and fungal suspension (80 μL, 106 CFU/ 25 °C. The wells were washed with water to remove mL) was added in the second well to the last well in unaccompanied violet crystal and subsequently the row. The third well (containing microorganisms the dye adhered to the biofilm was solubilized in the absence of the sample) corresponded to the in absolute ethanol (20 min) and the absorbance 100% growth control. The plates were incubated at was measured at 570 ηm. All experiments were 36 ºC and the optical density was measured at time performed in triplicate. zero and after 24 h of incubation using a microplate reader. The Minimal Inhibitory Concentration VIABILITY AND PROLIFERATION ASSAYS MIC90 and MIC50 corresponded to the lowest AGAINST MICE SPLENOCYTES concentration of the sample capable of promoting Animals a reduction of ≥90% or ≥50%, respectively, in optical density, as compared to the 100% Female BALB/c mice (6-8 weeks old; 5 animals) growth control. For determination of Minimum were raised and maintained at the animal facilities Bactericidal Concentration (MBC) and Minimum of the Keizo Asami Immunopathology Laboratory - Fungicidal Concentration (MFC), aliquots (10 LIKA located in Federal University of Pernambuco, μL) of the wells containing concentrations of Brazil. Mice were kept under standard laboratory ≥MIC50 samples were inoculated into petri dishes conditions (20-22 °C and 12 h day and night cycle) containing Mueller Hinton or Sabouraud Dextrose with free access to standard diet (Labina/Purina, agar medium, which were subsequently incubated Campinas, Brazil) and water. All experimental at 36 °C for 24h. The MBC and MFC corresponded procedures were performed according to the Ethics to the lower concentration of the sample able to Committee of Animal Use (CEUA) of Federal
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University of Pernambuco (protocol number: and Annexin V conjugated with fluorescein 0048/2016). isothiocyanate (FITC): 50 μg/mL) and propidium iodide (PI, 20 μg/mL) were added to each labeled Preparation of splenocytes cytometer tube. Flow cytometry was performed in a This procedure was performed in accordance with FACS Calibur (Becton Dickinson Biosciences) and Melo et al. 2011. After the inoculation of anesthetics analyzed using Cell Quest Pro software (Becton (10mg/Kg of xylazine and 115mg/Kg of ketamine Dickinson). Result of the analysis was performed i.p. injection) followed of cervical displacement, in graphs by dot plot. Annexin-FITC negative/PI spleen of each mouse was removed aseptically and positive cells were considered necrotic cells and placed in a Falcon tube containing RPMI 1640 with Annexin-FITC positive/PI negative represented fetal calf serum (complete medium). In a vertical splenocytes in the early stage of apoptosis. Double flow, each spleen was transferred to a petri dish negatives were considered viable cells. where they were soaked. The cell suspensions Cell proliferation analysis using CFSE staining obtained from each spleen were transferred to Falcon tubes containing approximately 10 mL of Same protocol to obtained splenocytes was incomplete medium. Spleen homogenates were used for proliferation assay. After splenocytes overlaid onto Ficoll-Paque™ PLUS layer, with the obtainment, cell solution was centrifuged at 300 x density adjusted to 1.076 g/mL, and centrifuged g at room temperature for 5 min with sterile PBS at 1000 x g at room temperature for 25 min. The 1X added with SFB 5% (pH 7.2). After this, the interface cell layer containing immune cells was cell solution was adjusted to10x106 cells/mL and recovered by Pasteur pipette, washed twice in received 5mM of 5(6)-carboxyfluorescein diacetate phosphate-buffered saline (PBS) and centrifuged N-succinimidyl ester (CFSE). Cells were incubated twice at 500 x g for 10 min. Cells were counted for 10 min at room temperature in the dark. Cells in a Neubauer chamber, and cell viability was were centrifuged twice at 300 x g / 5 min with sterile determined by the trypan blue exclusion method. PBS 1X. Cells stained were cultured for 24 and 48 Cells were only used when viability was> 98%. hours with 12.5 µg/mL of saline extract from M. emarginata leaves and only culture complete RPMI Analysis of cell viability by Annexin V-FITC and 1640 medium (negative control). After culture time Propidium Iodide Staining cells were centrifuged (300 x g / 5 min), were carry Mice splenocytes (106 cells) were treated with to acquisition on FACSCalibur platform (Becton saline extract from Malpighia emarginata leaves at Dickinson Biosciences) and results were analyzed 50, 25, 12.5, 6 and 3 μg/mL and were maintained using Cell Quest Pro software (Becton Dickinson). in 24-well plates for 24 hours to analyze their STATISTICAL ANALYSIS cell viability. Untreated cells (control) were used as a negative control. Following this, splenocytes To test the normal hypothesis on the variable were centrifuged at 4 °C, 450 x g for 10 min. After involved in this study the Shapiro–Wilke test was discarding the supernatant, 1 mL of PBS 1X was applied. Means of samples were analyzed using added to the precipitate and centrifuged at 4 °C, 450 non-parametric tests. The statistical difference x g for 10 min. After discarding the supernatant, between two groups was analyzed by Wilcoxon test the pellet was resuspended in a binding buffer of and that among more than three groups by one-way cell viability kit (Becton Dickinson Biosciences) analysis of variance (ANOVA). All the conclusions
An Acad Bras Cienc (2019) 91(1) e20180358 6 | 15 BÁRBARA R.S. BARROS et al. Malpighia emarginata PRESENT BIOLOGICAL ACTIVITIES were considered with a significance level of 5%. In addition, we found 18 compounds in the For statistical analysis was used GraphPad Prim saline extract in accordance with the area, retention 5.01 software. time, molecular weight, molecular formula and negative mass, identifying other phenolic RESULTS compounds, like Protocatechuic acid, Apigenin- 7-O- glucoside flavonoid and terpenoids, like PREVALENCE OF PHENOLIC COMPOUNDS IN Isotriptophenolide (see Table II). PHYTOCHEMICAL SCREENING OF SALINE EXTRACT FROM M. emarginata LEAVES SALINE EXTRACT FROM M. emarginata LEAVES SHOWED SIGNIFICANT ANTIOXIDANT POWER Results of analysis in UPLC-MS about the Saline extract from M. emarginata leaves showed phytochemical characterization of saline extract higher antioxidant power being five times superior from M. emarginata leaves (Figure 1) showed that to butylated hydroxytoluene (BHT) standard was found many phenolic compounds being some (20.32±0.89 / 4.12±0.10 to saline extract and compounds belonged flavonoid (Table I). BHT, respectively). Saline extract also was able to
Figure 1 - UPLC-MS Chromatogram of saline extract from M. emarginata leaves.
TABLE I Result of antioxidant activities and phenol amounts found on saline extract from M. emarginata leaves.
M. emarginata BHT Ascorbic acid
Total phenolic compounds (mg GAE/g) 51.14 ± 0.21 - - Total of flavonoids (mg QE/g) 9.67 ± 0.08 - - Free radical sequestration by DPPH (% mg/mL) 38.59 ± 1.20 93.13 ± 0.58 90.04 ± 0.16 Total antioxidant capacity (mg AAE/g) 20.32 ± 0.89 4.12 ± 0.10 100 Reduction of Ferric Ion (EFeSO4(II)/g) 416.11 ± 0.46 679.17 ± 25.98 1215 ± 48.55
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TABLE II UPLC-MS spectral analysis of compounds found in saline extract from M. emarginata leaves.
RETENTION MOLECULAR COMPOUNDS AREA MASS [M-H]- TIME (min) FORMULA
Rhinocerotinoic acid 0.63 C20H30O3 5.756.062 318.457 317.211
Quinic Acid 1.06 C7H12O6 105.468.063 192.063 192.063
Dimethoxycurcumin 2.72 C23H24O6 5.840.279 396.439 395.149
Protocatechuic Acid 3.36 C7H6O4 16.625.830 154.026 153.018
Tolypodiol 3.60 C28H40O5 6.356.449 456.623 455.279
Pauciflorol A 3.90 C20H34O2 73.426.766 306.255 305.248
Gentisic acid 4.25 C7H6O4 5.425.569 154.026 153.018
Matricin 4.38 C17H22O5 73.426.766 306.358 305.138
Gallocatechin 4.38 C15H14O7 7.342.677 306.27 305.066 11a-hidroxi-3,7-dioxo-5a-lanosta-8,24 4.49 C H O 7.038.835 484,318 483,311 (E)-dien-26-oic Acid 30 44 5
Protocatechuic acid 4.65 C7H6O4 5.425.569 154.121 153.018
Cicutoxin 5.19 C17H22O2 6.584.033 258.355 225.164
Salicylic acid 5.71 C7H6O3 31.334.580 138.031 137.023
2,5 Dihydroxybenzaldehyde 5.71 C7H6O3 31.334.580 138.031 137.023
Apigenin-7-O- glucoside 8.86 C21H20O10 6.690.703 432.105 431.097
Magnosalicin 8.87 C24H32O7 6.690.703 432.513 431.207
Apigenin-8-O- glucoside 8.87 C21H20O10 6.690.703 432.381 431.097
Isotriptophenolide 9.85 C20H24O3 50.121.777 312.409 311.164 increase of free radical sequestration (38,59 %) and concentrations (Table III). In addition, was also promoted higher ferric ion reduction, also like BHT possible observe that saline extract presented more standard (see Table I). significant results as antifungal against C. albicans and C. parapsilosis, comparing to the other species. HIGHER ANTIFUNGAL ACTIVITY WAS PROMOTED BY SALINE EXTRACT Only the C. glabrata species was resistant to saline extract. Six bacterial species tested showed resistance to Antibiofilm activity was evaluated with the the extract. However, the saline extract from M. saline extract following the concentrations of emarginata leaves inhibited 90% of growth capacity MICs: 64 MIC, 16 MIC, 4 MIC, MIC, 1/4 MIC, of Candida albicans, Candida parapsilosis, 1/16 MIC and 1/64 MIC; according to the graphs in Candida krusei and Candida tropicalis in lower figure 2 and Table IV.
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TABLE III Antifungal activity against Candida spp. Promoted by saline extract from Malpighia emarginata leaves.
Fluconazole Saline extract from M. emarginata leaves Strains (µg/mL) (µg/mL)
MIC50 MIC50 MIC90 MFC
C. albicans 0.25 0.51 0.52 ND
C. parapsilosis ND 0.51 0.51 0.51
C. krusei 32 8.36 8.36 ND
C. tropicalis 4 0.13 0.26 1.05
C. glabrata 64 ND ND ND
Figure 2 - Analysis of the antibiofilm activity ofMalpighia emarginata saline extract to species of Candida albicans (a), Candida krusei (b), Candida tropicalis (c) and Candida parapsilosis (d). Assays performed in triplicate. p < 0.002.
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TABLE IV Results on the antibiofilm activity of Malpighia emarginata saline extract, in percentage, of Candida ssp. tested.
Value% of growth (DO600) of Candida ssp. (mean) DO600 Control 64 MIC 16 MIC 4 MIC 1 MIC 1/4 MIC 1/16 MIC 1/64 MIC C. albicans 100 104.3 105.5 105.1 109.1 105.3 120.5 122.6 C. krusei 100 99 112.4 110.4 111.3 116.1 121.4 109.3 C. tropicalis 100 94 95.6 102.5 110.2 99.7 98.4 83.1 C. parapsilosis 100 118.7 116.4 112.2 109 104.3 96.6 116.8 Value% of biofilm biomass of Candida spp. (mean)
Control 64 MIC 16 MIC 4 MIC 1 MIC 1/4 MIC 1/16 MIC 1/64 MIC C. albicans 100 93.4 85.5 87.7 88.7 89.2 87.2 85.8 C. krusei 100 85 92.7 92 95.4 104.9 107 111.9 C. tropicalis 100 107.9 109.7 117.3 118 115 119,6 118.6 C. parapsilosis 100 90.5 85.3 74.3 77.2 70.4 77.5 73.3
The results of the antibiofilm activity confirm the antimicrobial tests performed. The saline extract of Malpighia emarginata presented a considerable reduction in the biofilm formation of C. albicans, C. krusei and C. parapsilosis species, as described in table IV.
SALINE EXTRACT FROM M. emarginata LEAVES NOT INDUCED CELL DAMAGE AND PROMOTED HIGHER CELL PROLIFERATION IN MICE SPLEEN CELLS Figure 3 - Mice splenocytes viability evaluated using annexin V and propidium iodide staining. Saline extract from Mice Balb/c spleen cells treated with saline extract M. emarginata leaves did not promoted cell death in none in different concentrations (50, 25, 2.5, 6 and 3 μg/ concentration. Horizontal bars represent the average of three mL), did not suffer significant cell death, for both independent experiments performed in duplicate. Results were evaluated in the significance level of 5%. apoptosis and necrosis, in none concentrations evaluated (Figure 3). In fact, saline extract from DISCUSSION M. emarginata leaves promoted a higher cellular viability, superior to 90% of surviving. For Popular culture collaborates with the indiscriminate measuring if saline extract was able to activate use of medicinal plants within the context of self- immune spleen cells, we performed other assay medication, requiring the study of its constituents, to investigate the proliferation index of these the mechanism of therapeutic action and the cells challenged with only one concentration identification of active principles responsible for of extract (12.5 μg/mL) during two days of cell the various biological activities reported by the culture. Results showed that saline extract from population (Ford et al. 2014). Here, we used a M. emarginata leaves induced higher proliferation saline extract made from M. emarginata leaves. indices in both times evaluated (Figure 4). The proposal was investigated if an extract of plant,
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compound used as standard (Pasa et al. 2016, Nascimento et al. 2017). We suggest that this antioxidant potential is due to higher amounts of phenolic compounds, previously described in several studies as antioxidants molecules (Zengin et al. 2018). Similar to our results, other studies also showed that plants rich in phenolic compounds have antioxidant potential as well as the extracts of Ficus beecheyana (Yen et al. 2018), Punica granatum (Russo et al. 2018) and Saccharum Figure 4 - Mice splenocytes proliferation index evaluated using CFSE staining. Saline extract from M. emarginata officinarum (Abbas et al. 2014). Moreover, leaves promoted proliferation in mice splenocytes in both Nascimento et al. (2017) analyzed the antioxidant times evaluated (24 and 48 hours of assay). Vertical white bars activity of Malpighia glabra fruits at various represent negative control cells (cells + culture medium) and stages of maturation, thus finding high levels of vertical gray bars represent saline extract from M. emarginata leaves at 12.5 µg/mL concentration. This assay was performed antioxidant activity and, consequently, the presence using three independent experiments performed in duplicate. of phenolic compounds. #P= 0.003. Organic extracts from various organs of M. emarginata presented potential antimicrobial made with standard physiological solution (NaCl activity against different pathogenic bacteria 0.15M), is cytotoxic to cells and microorganisms species as described by Silva (2014) and Motohashi and if this extract is also able to be antioxidant. et al. (2004). Although such solvents (hexane and In similarity to the results found by Wong et ethyl acetate) have high toxicity to microorganisms al. (2008) and Nowotarska et al. (2017), the saline (Yung et al. 2016), the indiscriminate use of these extract from leaves of M. emarginata, as described solvents is also associated with intoxication, either in Table II prepared in its phytochemicals 18 by ingestion or inhalation, causing serious damage characterized compounds, being a larger number of to the individual, such as severe lung injury and a class of phenolic and terpenoid compounds. hepatotoxicity (Ford et al. 2014, Tormohelen et al. Among the compounds described are the 2014, Connellan, 2017). apigenin-7-O-glucoside and Apigenin-8-C- The results obtained in this study did not glucoside flavonoids extensively studied and show action against the species of bacteria used. described in the literature for their potential However, the saline extract of M. emarginata applicability as anti-inflammatory agents (Kowalski leaves presented a potential fungicidal, fungistatic et al. 2005) and antitumor agents (Bankeu et al. and antibiofilm action, suggesting this capacity, 2017, Smiljkovic et al. 2017, Zhao et al. 2017). In once again, to the presence of phenolic compounds addition, Apigenin-8-C-glucoside is a biologically (Haghdoost et al. 2016, Barral et al. 2017, Jara et al. active molecule with potential antihypertensive, Wang et al. 2018). antidepressant, antispasmodic and antioxidant As described by Khan et al. (2014) and effect (Ele et al. 2016). Glorybai et al. (2015) plants are commonly used As described in Table I, saline extract from M. by traditional medicine as a source of cure for emarginata leaves showed a potential antioxidant various diseases. However, the irrational use or the capacity, especially when compared to butylated conception that a natural product does not cause hydroxytoluene (BHT) a known antioxidant harm to the health can be harmful to the individual,
An Acad Bras Cienc (2019) 91(1) e20180358 11 | 15 BÁRBARA R.S. BARROS et al. Malpighia emarginata PRESENT BIOLOGICAL ACTIVITIES being associated to diverse problems like poisoning capacity against colon cancer cell lines (HT29) and and anaphylactic shocks (Lin et al. 2018), being the hepatocellular carcinoma (HepG2) (Specian et al. toxicological studies of medicinal plants, of great 2016, Verdam et al. 2014). Our results associated relevance to the security of the individual (Saleh-e- with these findings suggest the possibility of future In & Staden, 2018). investigations about antitumor, wound healing Here we also investigate the cytotoxicity and immunological responses promoted by saline profile promoted by the saline extract from M. extract from Malpighia emarginata leaves. emarginata leaves in mice splenocytes and the results showed that, same in higher concentrations, CONCLUSIONS this extract is not toxic. Moreover, M. emarginata Saline extract from Malpighia emarginata leaves extract also promoted lymphocyte proliferation in showed the presence of a considerable concentration both 24 and 48 hours of incubation, suggesting of total phenolic compounds, among them some immunostimulatory properties. Recent study showed that extract of several species of Byrsonima flavonoids and phenolic acids. Moreover, showed spp. (Malpighiaceae) in different concentrations antioxidant and antifungal properties and was not (12.5 and 50 μg/mL) were not cytotoxic against cytotoxicity against mice splenocytes, inducing RAW 264-7 cells (Fraige et al. 2018). In 2016, high proliferation in these cells. These results Düsman et al. evaluated the cytotoxicity of the are promisors, demonstrate the safe use of this aqueous extract from Malpighia glabra Linnaeus plant against normal cells and stimulate new fruits in vivo and in vitro in Wistar rats, and against investigations to use this extract like antifungal and bone marrow derived cells from these animals. immunostimulant compound in future assays. Their results demonstrated that the extract also was ACKNOWLEDGMENTS not cytotoxic. The activation and cellular proliferation are The authors express their thanks to teacher Marlene directly associated with healing processes and with Barbosa, curator of Herbarium Geraldo Mariz, the replacement of dead cells of the tissues that are to the Center for Strategic Technologies of the in constant development in the organism. For this, Northeast (CETENE), to the Laboratory of Protein is very important investigate if a compound can Biochemistry; Biophysics; Biotechnological promote cell proliferation, especially if these cells Processes; Immunological and Antitumor Analyzes are immunological because the major activation and the Nucleus of Technological Platforms of the mechanism of lymphocytes is the clonal expansion Research Center Aggeu Magalhães (FIOCRUZ after it activation. Pernambuco). The authors declare no conflict of Plants extracts belonging to the Malpighiaceae interest. family also present other potential pharmacological properties, such as antimicrobial, wound healing AUTHOR CONTRIBUTIONS and antitumor (Düsman et al. 2016, Specian et al. 2016). B. Barros and B. Barboza performed the antioxidant, Recent studies with different species belonging antimicrobial and immunological assays, as well as to the genus Byrsonima (family Malpighiaceae) writing of the manuscript; B. Ramos and M. Moura showed no cytotoxic action against different human performed the antimicrobial assays; L. Coelho, cell lines (monocytes and cells of primary gastric T. Napoleão, M. Correia, P. Paiva and C. Lima epithelium) but demonstrated a potential cytotoxic made available different reagents and equipment
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